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EP3004118B1 - Use of condensed benzo[b]thiazine derivatives as cytoprotectants - Google Patents

Use of condensed benzo[b]thiazine derivatives as cytoprotectants Download PDF

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EP3004118B1
EP3004118B1 EP14734525.0A EP14734525A EP3004118B1 EP 3004118 B1 EP3004118 B1 EP 3004118B1 EP 14734525 A EP14734525 A EP 14734525A EP 3004118 B1 EP3004118 B1 EP 3004118B1
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pyrido
benzo
thiazin
trifluoromethoxy
dihydro
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French (fr)
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EP3004118A1 (en
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Jari Ratilainen
Gundars GOLDSTEINS
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Aranda Pharma Ltd
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Aranda Pharma Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/542—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/547—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame spiro-condensed or forming part of bridged ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D513/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00
    • C07D513/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00 in which the condensed system contains two hetero rings
    • C07D513/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D513/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00
    • C07D513/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00 in which the condensed system contains two hetero rings
    • C07D513/10—Spiro-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D513/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00
    • C07D513/12—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00 in which the condensed system contains three hetero rings
    • C07D513/20—Spiro-condensed systems

Definitions

  • lipid peroxidation is mediated through both enzymatic and non-enzymatic pathways.
  • Enzymatic pathways involve lipo-oxygenases and cyclo-oxygenases acting at the polyunsaturated fatty acids (PUFAs) present as free or engaged in lipid complexes such as cell membrane phospholipids or lipoproteins.
  • Non-enzymatic reactions on PUFAs (such as linoleic acid, arachidonic acid) involve ferryl radical, peroxynitrite, hydroperoxyl and hydroxyl radicals among others as possible mediators.
  • PUFAs such as linoleic acid, arachidonic acid
  • ferryl radical, peroxynitrite, hydroperoxyl and hydroxyl radicals among others as possible mediators.
  • Lipid hydroperoxides themselves are reactive oxygen species capable of oxidizing other macromolecules. Majority of the lipid hydroperoxides is, however, converted non-enzymatically to secondary products including electrophiles causing further dysfunction and damage in cells and leading to the acceleration of secondary lipid peroxidation and ultimately to cell death. Enhanced lipid peroxidation also contributes to the generation of inflammatory response and propagation of inflammatory processes in diseased tissues.
  • the cells employ various enzymatic and chemical reactions to defend against the formation and accumulation of lipid hydroperoxides.
  • the function of most of the cellular antioxidant systems is dependent on reducing equivalents such as glutathione, which is depleted upon aging and in early stages of pathogenic processes, rendering these defense mechanisms vulnerable.
  • An object of the present disclosure is to provide compounds useful as cytoprotectants and which may be used for the treatment or prophylaxis of peripheral and central degenerative disorders, in particular in the treatment of mammals, including humans.
  • US 4,861,878 discloses 1H-pyrido-[2,3-b][1,4]-thiazines, which are potent lipoxygenase inhibitors.
  • WO 2008/009935 discloses use of certain phenothiazine derivatives in treating disorders mediated by lipid peroxidation.
  • the present invention provides compounds of formula (I) for use as medicaments, in particular for use in the treatment of a condition where elimination of lipid hydroperoxides and/or limiting their detrimental effects on cellular macromolecules is desired.
  • the present invention further provides compounds of formula (I) for use in the treatment or prevention of diseases or states, either acute or chronic, involving aberrant cellular lipid peroxidation in the central nervous system or in the periphery of the body.
  • the present invention also provides novel arylthiazine compounds of formula (I).
  • the present invention further provides pharmaceutical compositions comprising one or more compounds of formula (I).
  • the present invention also provides a method for the preparation of novel compounds of formula (I).
  • the present invention relates to arylthiazine compounds having formula (I), wherein
  • the present invention relates to compounds of formula (I) wherein i is 0, i.e. having formula (I'). wherein G1, G2, G3, G4, G5, G6, G7, G8, R1, and m are as defined herein.
  • G7 is C(R7) 2 .
  • both R7 are C 1-6 -alkyl, such as methyl or ethyl, or methoxy-C 1-3 -alkylenyl; or form together with the ring carbon they are attached to a 3 to 7 membered aliphatic carbocyclic or heterocyclic ring optionally substituted one or two times with R5.
  • one R7 is H and the other R7 is as defined herein, in particular C 1-6 -alkyl,-[CH 2 ] k Ar, -[CH 2 ] k Cy, or C 1-3 -alkoxy-C 1-3 -alkylenyl.
  • Compounds of formula (I) can be used as medicaments, in particular in the treatment or prevention of diseases or states, either acute or chronic, involving aberrant cellular lipid peroxidation in the central nervous system or in the periphery of the body.
  • compositions of the present invention can be administered by any means that achieve their intended purpose.
  • administrations include, but are not limited to, parenteral, subcutaneous, intravenous, intraarticular, intrathecal, intramuscular, intraperitoneal, by intradermal injections, via transdermal, rectal, buccal, oromucosal, nasal, ocular routes, via inhalation, and via implant.
  • administration can be by the oral route.
  • C 2-6 -alkenyl refers to an unsaturated linear or branched hydrocarbon groups having one or more olefinic double bond between any two carbon atoms and containing suitably 2 to 6 carbon atoms, such as ethenyl, propenyl, butenyl, pentenyl, and hexenyl.
  • Preferred alkenyl groups of the present invention are linear alkenyl groups having a terminal double bond such as vinyl and allyl groups.
  • C 2-6 -alkynyl refers to an unsaturated linear or branched hydrocarbon group having at least one olefinic triple bond between any two carbon atoms, such as ethynyl, propynyl, butynyl, pentynyl, and hexynyl.
  • alkynyl groups include, but are not limited to, linear alkynyls groups having a terminal triple bond.
  • 5 or 6 membered saturated heterocyclic ring represents a stable 5 to 6 membered monocyclic ring and which consists of ring carbon atoms and from 1 to 4, preferably 1 to 2 in the case of saturated heterocyclic rings, heteroatom(s) each independently selected from a group consisting of N, O, and S, wherein N when applicable represents NH or may be otherwise further substituted.
  • the heterocyclic ring may be further substituted at any carbon atom or nitrogen heteroatom suitable for substitution, wherein the substituent is preferably hydroxyl, thiol, benzyloxy, or an aforedefined alkyl, more preferably methyl.
  • preferred saturated heterocyclic rings include, but are not limited to, pyrrolidinyl, piperidinyl, N-methyl piperidinyl, piperazinyl, N-methyl piperazinyl, and morpholinyl.
  • pharmaceutically acceptable salt includes any non-toxic organic and inorganic acid or base addition salts that compounds of formula (I) can form.
  • inorganic acids which form suitable salts, include, but are not limited to, hydrogen chloride, hydrogen bromide, sulphuric and phosphoric acids.
  • organic acids which form suitable salts, include, but are not limited to, acetic acid, lactic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, malic acid, tartaric acid, citric acid, ascorbic acid, maleic acid, benzoic acid, phenylacetic acid, cinnamic acid, methane sulfonic acid, salicylic acid, and the like.
  • Some of the compounds disclosed herein may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other steroisomeric forms.
  • the present invention is also meant to encompass racemic and/or steroisomeric mixtures, resolved forms, and mixtures thereof in all proportions, as well as the individual enantiomers and/or diastereomers that may be separated according to methods that are known to those skilled in the art.
  • the present invention is further meant to include any eventual metabolite, prodrug, and tautomeric forms of the compounds of the present invention.
  • Compounds of formula (IIb) can be obtained by reacting a compound of formula (V) wherein G1, G2, G3, G4, R1, and m are as defined above; with potassium thiocyanate to obtain a compound of formula (IV) and treating the obtained compound of formula (IV) with base, preferably KOH, to obtain a corresponding compound of formula (IIb) wherein G1, G2, G3, G4, R1, and m are as defined above.
  • N-Bromosuccinimide (67 mg, 0.37 mmol) was added to a solution of 2-chloro-4-methoxypyrimidin-5-amine (50 mg, 0.31 mmol) in chloroform (2 ml) and the resulting mixture was stirred at RT for 3 hours. Water was added and the mixture extracted with chloroform. The organic phase was washed with water and brine, dried over sodium sulphate and concentrated under reduced pressure. The yield of 4-bromo-2-chloro-6-methoxypyrimidin-5-amine was 60 mg.
  • Triethyl amine (9.36 ml, 67.26 mmol) was slowly added to a stirred mixture of ethyl 2-(4-aminooxan-4-yl)acetate hydrochloride (3.0 g, 13.45 mmol) in dichloromethane (60 ml).
  • Ethyl malonylchloride (1.9 ml, 14,79 mmol) was added dropwise at 0°C and the resulting mixture was allowed to warm up to RT and stirred at RT for 12 hours. After reaction was complete water was added. Organic phase was separated and the aqueous phase extracted with dichloromethane. The combined organic phases were washed with brine, dried over sodium sulphate and concentrated under reduced pressure.
  • Ethyl 3-amino-3-methylbutanoate hydrochloride was prepared according to the method described for ethyl 2-(4-aminooxan-4-yl)acetate hydrochloride.
  • Ethyl 3-methylbut-2-enoate (5 g, 29.4 mmol)
  • ethanol (20 ml)
  • liquid ammonia (20 ml) was used in the reaction.
  • the yield of ethyl 3-amino-3-methylbutanoate hydrochloride was 6.0 g.
  • Ethyl 3-(3-ethoxy-3-oxopropanamido)-3-methylbutanoate was prepared according to the method described for ethyl 2- ⁇ [4-(2-ethoxy-2-oxoethyl)-oxan-4-yl]carbamoyl ⁇ acetate.
  • Ethyl 3-amino-3-methylbutanoate hydrochloride (6.0 g, 33.14 mmol), ethyl malonylchloride (4.46 ml, 34.80 mmol), triethylamine (23 ml, 165.1 mmol) and dichloromethane (60 ml) was used.
  • Ethyl 3-amino-3-ethylpentanoate hydrochloride was prepared according to the method described for ethyl 2-(4-aminooxan-4-yl)acetate hydrochloride.
  • Ethyl 3-ethylpent-2-enoate (2.5 g, 16.0 mmol), ethanol (10 ml) and liquid ammonia (10 ml) was used in the reaction.
  • the yield of ethyl 3-amino-3-ethylpentanoate hydrochloride was 3.0 g.
  • Ethyl 3-(3-ethoxy-3-oxopropanamido)-3-ethylpentanoate was prepared according to the method described for ethyl 2- ⁇ [4-(2-ethoxy-2-oxoethyl)oxan-4-yl]carbamoyl ⁇ acetate.
  • Ethyl 3-amino-3-ethylpentanoate hydrochloride (3.0 g, 14.38 mmol)
  • ethyl malonylchloride (1.93 ml, 15.07 mmol
  • triethylamine (10 ml, 71.7 mmol) and dichloromethane (50 ml) was used.
  • Ethyl 2-(diethoxyphosphoryl)acetate (5.86 g, 28.15 mmol) was slowly added to the mixture of 60% sodium hydride (1.05 g, 28.15 mmol) in THF (20 ml) at 0°C. The mixture was allowed to warm up to RT and stirred for 1 hour. Cyclopentanone (2.1 ml, 23.77 mmol) was slowly added in THF (10 ml) and the resulting mixture was stirred at RT for 2 hours. Water was added and the mixture was extracted with ethyl acetate. The organic phase was washed with water and brine, dried over sodium sulphate and concentrated under reduced pressure. The yield of ethyl 2-[cyclopentylidene]acetate after flash chromatography (100-200 mesh size silica gel, 5% ethyl acetate in hexane) was 3.0 g.
  • Ethyl 2- ⁇ [1-(2-ethoxy-2-oxoethyl)cyclopentyl]carbamoyl ⁇ acetate was prepared according to the method described for ethyl 2- ⁇ [4-(2-ethoxy-2-oxoethyl)oxan-4-yl]carbamoyl ⁇ acetate.
  • Ethyl 3-(3-ethoxy-N-ethyl-3-oxopropanamido)-3-methylbutanoate was prepared according to the method described for ethyl 2- ⁇ [4-(2-ethoxy-2-oxoethyl)oxan-4-yl]carbamoyl ⁇ acetate.
  • the yield of ethyl 3-(3-ethoxy-N-ethyl-3-oxopropanamido)-3-methylbutanoate was 1.1 g.
  • Ethyl 2-[4-(ethylamino)oxan-4-yl]acetate hydrochloride was prepared according to the method described for ethyl 3-(ethylamino)-3-methylbutanoate hydrochloride.
  • 2M ethylamine in THF (30 ml) and ethanol (20 ml) was used.
  • the yield of ethyl 2-[4-(ethylamino)oxan-4-yl]acetate hydrochloride was 0.9 g.
  • the yield of ethyl 2- ⁇ [4-(2-ethoxy-2-oxoethyl)oxan-4-yl](ethyl)carbamoyl ⁇ -acetate was 0.45 g.
  • Ethyl 3-ethylpent-2-enoate (0.5 g, 3.20 mmol), 70% aqueous ethyl amine (10 ml) and ethanol (10 ml) was placed in a seal tube and heated at 90°C for 72 hours. After cooling to RT, water was added and the mixture was extracted with ethyl acetate. Organic phase was washed with water, dried over sodium sulphate and concentrated under reduced pressure: The yield of ethyl 3-ethylpent-2-enoate after flash chromatography (100-200 mesh size silica gel, 10% methanol in dichloromethane) was 50 mg.
  • Ethyl 3-(3-ethoxy-N-ethyl-3-oxopropanamido)-3-ethylpentanoate was prepared according to the method described for ethyl 2- ⁇ [4-(2-ethoxy-2-oxoethyl)oxan-4-yl]carbamoyl ⁇ acetate.
  • ethyl 3-ethyl-3-(ethylamino)pentanoate hydrochloride 50 mg, 0.25 mmol
  • ethyl malonylchloride 0.035 ml, 0.27 mmol
  • triethylamine 0.1 ml, 0.74 mmol
  • dichloromethane 5 ml was used.
  • the yield of Ethyl 3-(3-ethoxy-N-ethyl-3-oxopropanamido)-3-ethylpentanoate was 25 mg.
  • Oxane-3,5-dione ( III-5 ) was prepared according to the method described by Altenbach et al. in Journal of Medicinal Chemistry, 49(23), 6869-6887; 2006
  • Ethyl 3-(3-ethoxy-3-oxopropanamido)-2,2-dimethylpropanoate was prepared according to the method described for ethyl 2- ⁇ [4-(2-ethoxy-2-oxoethyl)oxan-4-yl]carbamoyl ⁇ acetate.
  • Ethyl 3-amino-2,2-dimethylpropanoate (0.7 g, 4.87 mmol), ethyl malonylchloride (0.68 ml, 5.31 mmol), triethylamine (2 ml, 14.48 mmol) and dichloromethane (10 ml) was used.
  • the yield of ethyl 3-(3-ethoxy-3-oxopropanamido)-2,2-dimethylpropanoate was 0.3 g.
  • Ethyl 2-[(4-ethoxy-2,2-dimethyl-3-oxobutyl)(ethyl)carbamoyl]acetate was prepared according to the method described for ethyl 2- ⁇ [4-(2-ethoxy-2-oxoethyl)oxan-4-yl]carbamoyl ⁇ acetate.
  • Ethyl 3-amino-3-phenylpropanoate hydrochloride was prepared according to the method described for ethyl 2-(4-aminooxan-4-yl)acetate hydrochloride.
  • Ethyl 3-phenylprop-2-enoate (3 g, 28.4 mmol)
  • ethanol (20 ml
  • liquid ammonia 25 ml
  • the yield of ethyl 3-amino-3-phenylpropanoate hydrochloride was 2.0 g.
  • 6-Phenylpiperidine-2,4-dione was prepared according to the method described for 9-oxa-1-azaspiro[5.5]undecane-2,4-dione ( III-15 ). Ethyl 3-(3-ethoxy-3-oxopropanamido)-3-phenylpropanoate (1.3 g, 4.28 mmol), Na-metal (0.15 g, 6.35 mmol), ethanol (5 ml), toluene (10 ml) and acetonitrile containing 1% of water (10 ml) was used. The yield of 6-phenylpiperidine-2,4-dione ( III-3 1) was 0.6 g.
  • 6-methylpiperidine-2,4-dione ( III-32 ) was prepared according to the same method as described for 6-phenylpiperidine-2,4-dione ( III-31 ). Ethyl but-2-enoate was used as a starting material.
  • Ethyl 3-[N'-(3-ethoxy-3-oxopropanoyl)acetohydrazido]-3-methylbutanoate was prepared according to the method described for ethyl 2- ⁇ [4-(2-ethoxy-2-oxoethyl)oxan-4-yl]carbamoyl ⁇ acetate.
  • Ethyl 3-acetohydrazido-3-methylbutanoate (1.5 g, 7.42 mmol), ethyl malonylchloride (1.05 ml, 8.16 mmol), triethylamine (1.55 ml, 11.1 mmol) and dichloromethane (30 ml) was used.
  • III-41 1-acetoamino-6,6-dimethylpiperidine-2,4-dione was prepared according to the method described for 9-oxa-1-azaspiro[5.5]undecane-2,4-dione ( III-15 ). Ethyl 3-[N'-(3-ethoxy-3-oxopropanoyl)acetohydrazido]-3-methylbutanoate (50 mg, 0.16 mmol), Na-metal (6 mg, 0.22 mmol), ethanol (1 ml), toluene (2 ml) and acetonitrile containing 1% of water (2 ml) was used. The yield of 1-acetoamino-6,6-dimethylpiperidine-2,4-dione ( III-41 ) was 12 mg.
  • N- ⁇ 2,4-dioxo-9-oxa-1-azaspiro[5.5]undecan-1-yl ⁇ acetamide ( III-66 ) was prepared according to the same method as described for 1-acetoamino-6,6-dimethylpiperidine-2,4-dione ( III-41 ). Ethyl 2-(oxan-4-ylidene)acetate was used as a starting material.
  • Ethyl 3- ⁇ N'-[1-(2-ethoxy-2-oxoethyl)cyclopentyl]acetohydrazido ⁇ -3-oxopropanoate was prepared according to the method described for ethyl 2- ⁇ [4-(2-ethoxy-2-oxoethyl)oxan-4-yl]carbamoyl ⁇ acetate.
  • III-78 1-acetamino-6-azaspiro[4.5]decane-7,9-dione was prepared according to the method described for 9-oxa-1-azaspiro[5.5]undecane-2,4-dione ( III-15 ). Ethyl 3- ⁇ N'-[1-(2-ethoxy-2-oxoethyl)cyclopentyl]acetohydrazido ⁇ -3-oxopropanoate (120 mg, 0.35 mmol), NaOEt (36 mg, 0.53 mmol), ethanol (0.5 ml), toluene (6 ml) and acetonitrile containing 1% of water (6 ml) was used. The yield of 1-acetamino-6-azaspiro[4.5]decane-7,9-dione ( III-78 ) was 55 mg.
  • Ethyl 2-(1-aminocyclopropyl)-2-methylpropanoate was prepared according to the method described by Bertus et al. in Synlett 2003(2), 265-267 .
  • Ethyl 2-cyano-2,2-dimethylacetate was used as a starting material.
  • Ethyl 2- ⁇ [1-(1-ethoxy-2-methyl-1-oxopropan-2-yl)cyclopropyl] carbamoyl ⁇ acetate was prepared according to the method described for ethyl 2- ⁇ [4-(2-ethoxy-2-oxoethyl)oxan-4-yl]carbamoyl ⁇ acetate.
  • Ethyl 2-(1-aminocyclo-propyl)-2-methylpropanoate (0.7 g, 4.09 mmol), ethyl malonylchloride (0.8 ml, 6.14 mmol), triethylamine (1.7 ml, 12.28 mmol) and dichloromethane (10 ml) was used.
  • the yield of ethyl 2- ⁇ [1-(1-ethoxy-2-methyl-1-oxopropan-2-yl)cyclopropyl]carbamoyl ⁇ acetate was 0.25 g.
  • III-81 .8,8-Dimethyl-4-azaspiro[2.5]octane-5,7-dione was prepared according to the method described for 9-oxa-1-azaspiro[5.5]undecane-2,4-dione ( 111-15 ).
  • the yield of 8,8-Dimethyl-4-azaspiro[2.5]octane-5,7-dione ( III-81 ) was 0.1 g.
  • Activated charcoal (15 mg) was added to the solution of Compound 32 (30 mg, 0.09 mmol) in acetic acid (2 ml). The resulting mixture was stirred at 120°C under oxygen atmosphere for 30 minutes. After cooling the mixture was filtered through celite pad and the filtrate was evaporated to dryness. Water was added and the mixture was neutralized with saturated aqueous Na-HCO 3 solution and extracted with dichloromethane. The organic phase was washed with brine, dried over sodium sulphate and concentrated under reduced pressure. The yield after preparative HPLC was 3.5 mg.
  • Compound 59 was prepared according to the same procedure used for the preparation of Compound 34.
  • Compound 57 (30 mg, 0.08 mmol) was used as starting material.
  • the yield after preparative HPLC was 3.0 mg.
  • Methyl iodide (37 mg, 0.26 mmol) was added to the mixture of Compound 43 (30 mg, 0.087 mmol) and K 2 CO 3 (2 mg, 0.104 mmol) in DMF (1 ml). The resulting mixture was stirred at RT for 16 hours. Brine was added to the mixture and the resulting mixture was extracted with ethyl acetate. The organic phase was dried over sodium sulphate and concentrated under reduced pressure. The yield after preparative-TLC (2% methanol in dichloromethane) was 4 mg.
  • Methyl iodide (37 mg, 0.26 mmol) was added to the mixture of Compound 43 (30 mg, 0.087 mmol) and K 2 CO 3 (2 mg, 0.104 mmol) in DMF (1 ml). The resulting mixture was stirred at RT for 16 hours. Brine was added to the mixture and the resulting mixture was extracted with ethyl acetate. The organic phase was dried over sodium sulphate and concentrated under reduced pressure. The yield after preparative-TLC (2% methanol in dichloromethane) was 6 mg.
  • Propargyl bromide (10 mg, 0.08 mmol) was added to a stirred mixture of compound 61 (15 mg, 0.04 mmol) and K 2 CO 3 (7 mg, 0.05 mmol) in DMF (1 ml) at RT. The resulting mixture was stirred at RT for 4 hours. The mixture was poured in an ice water and extracted with ethyl acetate. The organic phase was washed with water, dried over sodium sulphate and concentrated under reduced pressure. The yield after preparative TLC (30% ethyl acetate in hexane) was 2.5 mg.
  • Methyl iodide (44 mg, 0.31 mmol) was added to the mixture of Compound 67 (40 mg, 0.103 mmol) and K 2 CO 3 (21 mg, 0.155 mmol) in DMF (1 ml). The resulting mixture was stirred at RT for 4 hours. Brine was added to the mixture and the resulting mixture was extracted with dichloromethane. The organic phase was dried over sodium sulphate and concentrated under reduced pressure. The yield after preparative-TLC (30% ethyl acetate in hexane) was 1 mg.
  • Methyl iodide (44 mg, 0.31 mmol) was added to the mixture of Compound 67 (40 mg, 0.103 mmol) and K 2 CO 3 (21 mg, 0.155 mmol) in DMF (1 ml). The resulting mixture was stirred at RT for 4 hours. Brine was added to the mixture and the resulting mixture was extracted with dichloromethane. The organic phase was dried over sodium sulphate and concentrated under reduced pressure. The yield after preparative-TLC (30% ethyl acetate in hexane) was 3 mg.
  • the compounds of the present invention limit the damage caused by lipid hydroperoxides, act as cytoprotectants in various cell death models in vitro, suppress the production of inflammatory mediator and provide protection against neurotoxin in vivo. Said properties are demonstrated with the pharmacological experiments described below.
  • Undifferentiated PC12 cells were plated in 96-well plates at the density of 10,000 cells/well in 100 ⁇ l medium (Dulbecco's Modified Eagle Medium GlutaMAX Gibco, supplemented with 5% fetal bovine serum and 5 % horse serum). Medium was replaced 48 h after plating with 100 ⁇ l serum-free medium containing various concentrations of LOOH to first establish dose response for LOOH induced cell death.
  • concentration of LOOH that yielded 70-90% cytotoxicity was selected for screening potential cytoprotective effects of compounds of the present invention.
  • Compounds were first dissolved in 100% DMSO at 10mM and then diluted with culture medium to appropriate working solutions. Compounds were studied at concentrations of 0, 80 nM, 400 nM, 2000 nM, and 10 000 nM. Compounds were plated on cells at the same time as LOOH was added. Controls included cells exposed to plain medium (scaled to 100% viability), LOOH only, or study compounds alone without LOOH to assess potential cytotoxicity of the study compounds on PC12 cells. After 24-h incubation resazurin viability assay was performed.
  • Resazurin is a dye producing highly fluorescent resorufin when reduced by oxidoreductases within viable cells. Measurement of resazurin fluorescence is therefore an indicator of the viability of the cell. Following the LOOH exposure, medium was removed and replaced with 100 ⁇ l of 10 ⁇ M pre-warmed resazurin (Sigma). The working solution of resazurin was prepared from 50 mM resazurin in Hank's Buffered Salt Solution stock solution. The plates were incubated for 2 h at 37°C, 5% CO 2 . Resorufin fluorescence was measured at 530 nm / 590 nm (excitation/emission) using Victor 1420 multilabel reader.
  • Student's T-test was used to determine whether the difference between the means for two measurement groups (treatment group at selected concentration vs. vehicle treated cells) was statistically significant.
  • ROS Reactive oxygen species
  • RNS reactive nitrogen species
  • PC12 cells were therefore exposed to a combined ROS/RNS injury in order to generate a model system for studying efficacies of compounds capable of limiting lipid hydroperoxide cytotoxicity.
  • Cell culture methods similar to Experiment 1 were used except that a combination of superoxide donor (paraquat, PQ, Aldrich) and nitric oxide releasing sodium nitroprusside (SNP, Sigma) was used as toxin.
  • Paraquat and SNP both at 100 ⁇ M final concentration were mixed and plated on PC12 cells at the same time with study compounds. Resazurin viability assay was performed 24 h later as described above.
  • the murine microglial BV2 cell line was grown in RPMI-1640 medium containing 10% heat inactivated FBS supplemented with L-glutamine at 4 mM final concentration and 5 ⁇ g/ml of gentamicin at 37°C in a humidified atmosphere of 5% CO 2 .
  • Cultured BV2 cells were then stimulated with 50 ng/ml LPS (Sigma) for 24 h in the absence or presence of various concentrations (2.5 ⁇ M, 5 ⁇ M, 10 ⁇ M, and 20 ⁇ M) of study compounds.
  • NO release was analyzed as nitrite, which is the primary stable and nonvolatile breakdown product of NO, by Griess reagent system (Promega) according to manufacturer's instructions.
  • the viability of BV2 cells as assessed by resazurin assay as described in Experiment 1, in the presence of increasing concentrations of study compounds was noted to be unchanged.
  • the NO release from BV2 cells exposed to LPS only was normalized to 100%.
  • MPP+ 1-methyl-4-phenyl-pyridinium
  • DA dopaminergic
  • Transgenic C. elegans integrated strain Is11-7 [Pdat-1:GFP] expressing GFP in its genome under the control of dopamine transporter-1 gene were cultivated and maintained according to the standard protocol. Strain was thawed from freezer vial and placed on freshly prepared Nematode Growth Medium (NGM) that have been spread with Escherichia coli ( E.coli ) strain OP50 as a food source and allowed to reproduce for several days. Hermaphrodite form of this C.elegans strain has eight DA neurons, six located in the head and two in the tail.
  • the culture was synchronized.
  • the NGM plates containing a layer of E. coli OP50 were inoculated with 4-5 drops of L1 worms from previous week experiments and incubated at 15°C for 4 days. Growth of nematode strains was daily followed with a stereomicroscope (Nikon SMZ645).
  • the eggs carrying adult worms were bleached and the eggs were used for the experiment and also for maintenance of the strain.
  • the eggs for the experiment were grown in liquid medium at 20°C on rotatin g shaker at 350 rpm for 14 h to develop to L1 stage.
  • the eggs used for maintenance were stored at 15°C in incubator up to 2 weeks.
  • a suspension of L1 worms and defined amount of E . coli strain OP50 in NGM was distributed into 96-well plates.
  • a 40- ⁇ l suspension contained an average of 40 animals per well.
  • MPP+ (Sigma) was established.
  • MPP+ ranging from 0 to 2mM concentration (in sterile filtered aqua as the total volume of 50 ⁇ l/well) was added to the plates and incubated at 20°C for 48 h in humidity chamber, and then analyzed.
  • a selected MPP+ concentration (0.75 mM) in the presence or absence of study compounds was added to the plates and incubated at 20°C for 48 h in humidity chamber on rotating shaker.
  • the compounds were prepared as stock solutions in DMSO, and then diluted to working solutions at the desired concentrations to yield a final volume of 50 ⁇ l aqua per well.
  • worms were anaesthetized by addition of sodium azide to a concentration of 14 mM. Plates were imaged with a BD pathway 855 High-Content Imager (Becton Dickinson Biosciences) with 10x objective acquiring 25 contiguous fields per well under control of Attovision software (Becton Dickinson Biosciences). Number of worms analyzed per treatment group was approximately 200.
  • the worms were mounted on glass slides to be imaged under 20x objective with a conventional fluorescence microscope (Olympus AX70) attached to Olympus Soft Imaging system. In this case 15 worms per treatment group were analyzed and each individual experiment was repeated 3 to 4 times.
  • GFP fluorescence derived from DA neurons was analyzed either with Attovision software or Image Pro Plus (Media Cybernetics). The data were normalized to the average of GFP fluorescence derived from vehicle treated C. elegans.
  • the cytoprotective properties of the compounds of the present invention are demonstrated using in vitro model systems in which antioxidant reserves such as glutathione is significantly reduced or depleted by high doses of glutamate or other disease relevant insults, or toxicities are studied in other cell lines or primary cell cultures and standard in vivo models.
  • the properties of the compounds are also studied in relevant in vitro and in vivo tests for stability, permeability, pharmacological selectivity and specificity, safety, tolerability or toxicity.

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